Sending proteins to the lysosome for degradation is one of the chief mechanisms cells use to control the activity of cell-surface proteins. Ubiquitination of membrane proteins serves as a major signal for their sorting to lysosomes. The post-translational ligation of ubiquitin (Ub) to target proteins initiates their internalization from the cell surface and their transport into lumenal vesicles of multivesicular endosomes/multivesicular bodies (MVBs). A series of ESCRTs (Endosomal Sorting Complex Required for Transport: e.g. ESCRT-0,I,II,III)) are known to coordinate the sorting of ubiquitinated membrane protein cargo (Ub-cargo) with the formation of MVB lumenal vesicles and thus play critical roles in the process of lysosomal degradation. Yet, how ESCRT proteins recognize and move Ub-cargo as well as how they might participate in coordinating the activity of Ub ligases and Ub peptidases to modify and thus regulate the fate of Ub-cargo are unclear. The work proposed here has two main objectives: The first is to establish which proteins serve as endosomal Ub sorting receptors, and how they move Ub-cargo in coordination with their other functions. The second objective centers on the emerging concept that since ubiquitination is dynamic, there are many instances in which Ub ligases and peptidases can compete for the final disposition of cargo. We now propose that other protein complexes that work in parallel as ESCRT-0- like proteins, as well as other endosomal sorting complexes, associate with DUbs and ligases to acutely influence the sorting fate of specific Ub-cargo. The Specific Aims are to: Investigate the roles of Ub-binding by ESCRT-I and -II. Test the function of alternate ESCRT-0-like complexes. Test the role of Deubqiutinating enzymes in recycling proteins from endosomes PUBLIC HEALTH RELEVANCE: Failure to target particular membrane proteins to lysosomes leads to inappropriately hyperactive channels, transporters, or signaling receptors, which in turn contribute to a number of diseases including cancer, metabolic disorders, developmental abnormalities, heart disease, and hypertension. The work proposed here will investigate the biochemical and cell biological processes that ensure proper recognition and sorting of membrane proteins that are marked by ubiquitin for degradation in lysosomes. We believe that such understanding will open new avenues for therapeutic interventions that manipulate the process of lysosomal degradation to target a range of biological processes.